Abstract

The mRNA encoding a putative human enzyme named Epidermal Retinol Dehydrogenase 2 (RDH-E2) was found to be significantly elevated in psoriatic skin [Y. Matsuzaka, K. Okamoto, H. Tsuji, T. Mabuchi, A. Ozawa, G. Tamiya, H. Inoko, Identification of the hRDH-E2 gene, a novel member of the SDR family, and its increased expression in psoriatic lesion, Biochem. Biophys. Res. Commun. 297 (2002) 1171-1180]. This finding led the authors to propose that RDH-E2 may be involved in the pathogenesis of psoriasis through its potential role in retinoic acid biosynthesis and stimulation of keratinocyte proliferation. However, enzymatic activity for RDH-E2 has never been demonstrated. RDH-E2 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins, and is most closely related to the group of SDRs comprised of both NAD(+)- and NADP(+)-dependent enzymes with activities toward retinoid and steroid substrates. In this study, we began the characterization of RDH-E2 protein in order to determine whether it might play a role in retinoic acid biosynthesis. The results of this study show that, similarly to other SDR-type retinol dehydrogenases, RDH-E2 appears to be associated with the membranes of endoplasmic reticulum. Furthermore, RDH-E2 expressed in Sf9 insect cells as a fusion to the C-terminal His(6)-tag and purified using Ni(2+)-affinity chromatography recognizes all-trans-retinol and all-trans-retinaldehyde as substrates and exhibits a strong preference for NAD(+)/NADH as cofactors. Specific activity of RDH-E2 toward all-trans-retinoids is much lower than that of other retinoid-active SDRs, such as human RoDH4 or RDH10. The preference for NAD(+) suggests that RDH-E2 is likely to function in the oxidative direction in vivo, further supporting its potential role in the oxidation of retinol to retinaldehyde for retinoic acid biosynthesis in human keratinocytes.

HEK293 cells were transfected with either empty vector (A) or RDH-E2 expression vector (B) and incubated with anti-FLAG antibodies. RDH-E2 expression was visualized using FITC-conjugated antibodies. Endoplasmic reticulum of HEK293 cells was visualized by staining with FITC-conjugated concanavalin A (C).

Sf9 cells (105) were titrated with recombinant baculovirus carrying the expression construct for RDH-E2/His6. SDS-PAGE analysis of cell lysates revealed the presence of a new protein band of expected molecular mass in baculovirus infected cells (A). This band was recognized by antibodies against His-tag (B). Fractionation of Sf9 cells by differential centrifugation followed by Western blot analysis showed that RDH-E2 was localized in the 105,000 g pellet and 10,000 g pellet fractions of the cells (C). P, pellet; S, supernatant.

Forty to eighty micrograms of purified RDH-E2 were incubated with all-trans-retinol (dark grey) in the presence of NAD(P)+ or with all-trans-retinaldehyde (light grey) in the presence of NAD(P)H (A). The effect of additional imidazole on RDH-E2 was tested by comparing its activity toward all-trans-retinol in the absence (dark grey) or presence (patterned dark grey) of 200 mM imidazole added directly to the reaction mixture (B). RDH-E2/His6 fraction eluted with 250 mM imidazole off of the Ni2+-affinity resin was desalted using gel-filtration column. The protein was not lost as a result of desalting, as shown by SDS-PAGE (C), while the activity decreased significantly.

The gene encoding putative RDH-E2-similar (RDH-E2-s) protein was found to be localized next to RDH-E2 gene on chromosome 8 (A). Protein sequence alignment revealed that RDH-E2-similar is seven amino acids longer than RDH-E2 and has an Asp residue in the position (a.a. 67, bold) that determines cofactor specificity, suggesting a preference for NAD+ as a cofactor.